The Single-Molecule Approach to Membrane Protein Stoichiometry

Area of Science:

• Biophysics
• Molecular Biology
• Cell Biology

Background:

• Single-molecule imaging enables novel biological experiments.
• Determining subunit stoichiometry is crucial for understanding membrane protein function.

Purpose of the Study:

• To apply single-molecule imaging to ascertain the stoichiometry of the membrane protein prestin.
• To develop a method for quantifying membrane protein subunits.

Main Methods:

• Coupling prestin to enhanced green fluorescent protein (eGFP).
• Synthesizing prestin-eGFP in human embryonic kidney (HEK) cells.
• Preparing membrane fragments via osmotic lysis.
• Observing stepwise fluorescence decrease due to single fluorophore photobleaching.
• Counting photobleaching steps to deduce stoichiometry using a binomial model.

Main Results:

• Fluorescence of individual prestin-eGFP molecules decreased in discrete, equal-amplitude steps.
• The number of photobleaching steps per molecule was quantifiable.
• This stepwise decrease is characteristic of single fluorophore photobleaching.

Conclusions:

• Single-molecule imaging provides a robust method for determining membrane protein stoichiometry.
• The study successfully deduced the molecular stoichiometry of prestin.